Functional characterization of ProSAAS: similarities and differences with 7B2.

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroendocrinology

Background:

• Prohormone convertases (PCs) 1 and 2 are key enzymes in neuroendocrine tissues, cleaving peptide precursors.
• Endogenous binding proteins, 7B2 for PC2 and proSAAS for PC1, have been identified.
• 7B2 is crucial for PC2 maturation and activity, while its inhibitory role is also noted.

Purpose of the Study:

• To investigate if proSAAS affects PC1 beyond simple inhibition.
• To compare the functional similarities and differences between 7B2 and proSAAS.
• To elucidate the role of proSAAS in PC1 activity and peptide processing.

Main Methods:

• In vitro assays using recombinant proSAAS N-terminal domain (proSAAS-(1-180)) to assess PC1 stabilization and thermal denaturation.
• Transient and stable transfection of HEK 293 and CHO/PC1 cells with proSAAS cDNA variants to measure PC1 activity and mass.
• Stable transfection of AtT-20 cells with SAAS cDNAs to study proSAAS function in a neuroendocrine context, analyzing proopiomelanocortin and proenkephalin processing.

Main Results:

• ProSAAS-(1-180) did not stabilize PC1 activity or protect against thermal denaturation in vitro.
• Co-expression of proSAAS variants (proSAAS-(1-225) and proSAAS-(1-180)) reduced detectable PC1 activity, with increased PC1 mass suggesting inactivation.
• In AtT-20 cells, proSAAS-(1-225) slowed convertase-mediated processing of peptide precursors, but did not alter final processed peptide levels.

Conclusions:

• ProSAAS exhibits distinct intracellular interactions with PC1 compared to 7B2's interaction with PC2.
• ProSAAS, particularly its N-terminal domain, may mediate PC1 inactivation rather than stabilization.
• While both proSAAS and 7B2 inhibit PCs, their functional roles and mechanisms of action differ significantly.